This invention is in the field of tumor suppressor genes (anti-oncogenes) and relates in general to products and methods for practicing broad-spectrum tumor suppressor gene therapy of various human cancers. In particular, the invention relates to methods for treating tumor cells by: (1) administering vectors comprising a nucleic acid sequence coding for the novel proteins referred to herein as prostate tumor suppressor gene products (PTSG products); or, (2) administering an effective amount of a protein coded for by the nucleic acid sequence. The invention also relates to diagnosis of certain cancers such as prostate and colon cancer using the cloned nucleic acids of this invention.
Cancers and tumors are the second most prevalent cause of death in the United States, causing 547,000 deaths per year. One in three Americans will develop cancer, and one in five will die of cancer (Scientific American Medicine, part 12, I, 1, section dated 1987). While substantial progress has been made in identifying some of the likely environmental and hereditary causes of cancer, the statistics for the cancer death rate indicate a need for substantial improvement in the therapy for cancer and related diseases and disorders.
A number of so-called cancer genes, i.e., genes that have been implicated in the etiology of cancer, have been identified in connection with hereditary forms of cancer and in a large number of well-studied tumor cells. Study of cancer genes has helped provide some understanding of the process of tumorigenesis. While a great deal more remains to be learned about cancer genes, the known cancer genes serve as useful models for understanding tumorigenesis.
Cancer genes are broadly classified into "oncogenes" which, when activated, promote tumorigenesis, and "tumor suppressor genes" which, when damaged, fail to suppress tumorigenesis. While these classifications provide a useful method for conceptualizing tumorigenesis, it is also possible that a particular gene may play differing roles depending upon the particular allelic form of that gene, its regulatory elements, the genetic background and the tissue environment in which it is operating.
One widely considered working hypothesis of cancer is as follows: (1) Most of all human cancers are genetic diseases and (2) they result from the expression and/or failure of expression of specific genes (i.e. mutant versions of normal cellular growth regulatory genes or viral or other foreign genes in mammalian cells that cause inappropriate, untimely, or ectopic expression of other classes of vital growth-regulatory genes.
A simplistic view of the biologic basis for neoplasia is that there are two major classes of cancer genes. The first class consists of mutated or otherwise aberrant alleles of normal cellular genes that are involved in the control of cellular growth or replication. These genes are the cellular protooncogenes. When mutated, they can encode new cellular functions that disrupt normal cellular growth and replication. The consequence of these changes is the production of dominantly expressed tumor phenotypes. In this model of dominantly expressed oncogenes, a view that has predominated since the emergence of the concept of the genetic and mutational basis for neoplasia, it is imagined that the persistence of a single wild-type allele is not sufficient to prevent neoplastic changes in the developmental program or the growth properties of the cell. The genetic events responsible for the activation of these oncogenes therefore might be envisioned as "single-hit" events. The activation of tumorigenic activities of the myc oncogene in Burkitt lymphoma, the expression of bcr-abl chimeric gene product in patients with chronic myelogenous leukemia, the activation of the H-ras and K-ras oncogenes in other tumors represent some of the evidence for the involvement of such transforming oncogenes in clinical human cancer. An approach to genetic-based therapy for dominantly expressed neoplastic disease presumably would require specific shutdown or inactivation of expression of the responsible gene.
Tumor suppressor genes PA0 Multiple Steps and Oncogenetic Cooperation
A more recently discovered family of cancer-related genes are the so-called tumor-suppressor genes, sometimes referred to as antioncogenes, growth-suppressor, or cancer-suppressor genes. Recent research suggests strongly that it is loss-of-function mutations in this class of genes that is likely to be involved in the development of a high percentage of human cancers; more than a dozen good candidate human tumor-suppressor genes have been identified in several human cancers. The tumor suppressor genes involved in the pathogenesis of retinoblastoma (RB), breast, and other carcinomas (p53), Wilm's tumors (wt 1, 2) and colonic carcinoma (DCC) have been identified and cloned. Some aspects of their role in human tumorigenesis have been elucidated.
The retinoblastoma gene (RB) is the prototype tumor suppressor. The RB gene encodes a nuclear protein which is phosphorylated on both serine and threonine residues in a cell cycle dependent manner (Lee et al., Nature, 329:642-645 (1987); Buchkovich et al., Cell, 58:1097-105 (1989); Chen et al., Cell, 58:1193-1198 (1989); DeCaprio et al., Cell, 58:1085-1095 (1989)). The molecular mechanisms by which RB participates in these cellular activities has not been completely elucidated. A current model holds that RB interacts with many different cellular proteins and may execute its functions through these complexes. If the function of RB protein is to maintain cells at G0/G1 stage, RB must "corral" and inactivate other proteins which are active and essential for entering G1 progression (Lee et al., CSHSOB, LVI:211-217 (1991)). This "corral" hypothesis is consistent with recent observations that an important growth-enhancing transcriptional factor, E2F-1, is tightly regulated by Rb in a negative fashion (Helin et al., Cell, 70:337-350 (1992); Kaelin et al., Cell, 70:351-364 (1992); Shan et al., Mol. Cell. Biol., 12:5620-5631 (1992); Helin et al., Mol. Cell. Biol., 13:6501-6508 (1993); Shan et al., Mol. Cell. Biol., 14:229-309 (1994)). The instantly disclosed protein, PTSG, binds to the Rb protein and thus participation in the regulation of mitosis.
The familial breast cancer gene, BRCA-1, has been mapped at chromosome 17 q21-22 by linkage analysis. It is not clear whether this gene will behave as a tumor suppressor or dominant oncogene. However, the gene involved in human familial cancer syndrome such as Li-Fraumeni syndrome, p53, apparently acts as the classical tumor suppressor; similarly, the loss of RB gene is associated with hereditary retinoblastoma (Knudson, 1993, supra).
Between these two extreme pictures of transforming oncogenes and purely recessive tumor-suppressor genes lie a number of additional mechanisms apparently involved in the development of neoplastic changes characteristic of many human tumors. It has been assumed for many years that most human cancers are likely to result from multiple interactive genetic defects, none of which alone is sufficient but all of which are required for tumor development to occur. The true roles of both the cellular protooncogenes and the growth-regulating tumor-suppressor genes in neoplasia of mammalian cells are thought to represent a complex set of interactions between these two kinds of genes.
One current theory of carcinogenesis is that for some tumorous pathologies like adenocarcinoma of the prostate, oncogenesis occurs through the selection of several genetic changes, each modifying the expression or function of genes controlling cell growth or differentiation (Nowell, P. C., Science 194:23-28 (1976); Weinberg, R., Cancer Res. 49:3713-3721 (1989)). Even though adenocarcinoma of the prostate is ranked first in incidence and second in mortality among neoplasms in men (Coffey, D. S., Cancer 71:880-886 (1993)), little is known of the molecular basis of this common disease. For example, genetic alterations in colon cancer have been extensively studied and a model has been proposed in which the activation of oncogenes and loss of function of tumor suppressor genes is correlated with progressive clinical and histopathological changes observed during colorectal carcinogenesis (Fearon, E. R. and Vogelstein, B., Cell 61:759-767 (1990)). Indeed, a similar process of progressive genetic changes has been suggested to occur in prostate cancer (Isaacs, W. B. and Carter, B. S., Cancer Surveys, vol. 11, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 15-24 (1991)) but the exact location and mechanism of underlying genetic alteration remains unknown.
Known cancer genes have been shown not to be primarily responsible for prostate cancer. For example, mutations of cancer genes such as ras oncogenes or the tumor suppressor gene p53 have been found in only a small fraction (&lt;10%) of early prostatic tumors (Carter et al., Proc. Natl. Acad. Sci. U.S.A., 87:8751-8755 (1990); Gumerlock et al., Cancer Res. 51:1632-1637 (1991); Bookstein et al., Cancer Res. 53:3369-3373 (1993)); however, mutations of the latter have been detected in 20-25% of late-stage primary tumors, suggesting that the p53 gene can participate in one of several alternative pathways of prostate tumor progression (Bookstein et al., Cancer Res. 53:3369-3373 (1993)).
Karyotyping and allelotyping of tumor cells also has been used to try to find the genetic mechanisms responsible for prostate cancer. Cytogenetic studies of short-term cultures of primary prostatic cancers have disclosed several consistent chromosomal aberrations such as deletion of chromosomes 1p, 7q, or 10g (Atkin, N. B. and Baker, M. C., Hum. Genet., 70:359-364 (1985); Gibas et al., Cancer Genet. Cytogenet. 16:301-304 (1985); Lundgren et al., Genes Chrom. Cancer, 4:16-24 (1992)), whereas studies of allelic loss have suggested a somewhat different set of frequently lost chromosomal regions. Carter et al., Proc. Natl. Acad. Sci. U.S.A. 87:8751-8755 (1990), first reported non-random losses of chromosomes 10g and 16q each in .about.30% of 28 tumors, and Kunimi et al., Genomics 11:530-536 (1991), showed losses of these same regions as well as of the p arms of chromosomes 8 and 10 at rates exceeding 50% in their set of 18 tumors.
Allelic loss of chromosome 8p is detected in 65% of prostate carcinomas, the highest rate of any chromosome arm. These rates compare to those of allelic losses of Rb in retinoblastoma, 100% of which have Rb mutation, and suggest the inactivation of a tumor suppressor gene in 8p. Interestingly, karyotypic deletion of 8p has been noted in androgen-unresponsive sublines of cell line LNCaP. No previously cloned suppressor genes are located in 8p.
In the study of Bergerheim et al. (Bergerheim et al., Genes Chromosom. Cancer 3:215-220 (1991)), alleles of the NEFL locus on chromosome 8p12-p22 were lost from tumors in 7 out of 8 informative patients, and those of the lipoprotein lipase locus (8p22) were lost in 6 out of 7 patients. Alleles of the PLAT locus (8p12-q11) were retained in some tumors losing more distal 8p loci, implying that the putative suppressor locus is located on 8p distal to PLAT. The most distal marker, D8S7, was lost in 3 out of 6 tumors. The exceptionally high rates of allelic loss of LPL and NEFL, and the failure to observe allelic losses starting distal to these loci, further suggested that the suppressor locus may be relatively close to LPL or NEFL.
Thus, in order to effectively diagnose susceptibility to prostate cancer and related pathologies and other related cancers, and for treatment, the locale of a tumor suppressor gene responsible for these pathologies must be identified and located. This invention satisfies this need and provides related advantages as well.